Use of melanin biosynthesis inhibitors from korean ginseng and the cosmetic composition containing thereof for skin whitening

ABSTRACT

Disclosed herein is a whitening cosmetic composition containing plant-derived ginsenoside Fl (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) as an active ingredient. More specifically, the ginsenoside Fl is obtained from particularly a ginseng extract using an acid, a base, an enzyme or microorganism, and a whitening cosmetic composition containing the ginsenoside Fl has an excellent effect of inhibiting melanin biosynthesis, and thus provides an excellent skin whitening effect.

TECHNICAL FIELD

The present invention relates to a use of ginsenoside Fl obtained from aginseng extract using an acid, a base, an enzyme or a microorganismproducing the enzyme, and of a cosmetic composition containing the same,for skin whitening.

BACKGROUND ART

Various factors are involved in determining human skin color, and amongthem, factors, such as the activity of melanocytes, which make melaninpigments, the distribution of blood vessels, the thickness of the skin,and the presence or absence of pigments (e.g., carotenoid, bilirubin,etc.) in the human body, are of importance. The most important factoramong them is black pigment melanin, which is produced by the action ofvarious enzymes such as tyrosinase in human melanocytes. The formationof the melanin pigment is influenced by genetic factors, hormonesecretion, physiological factors associated with stresses, andenvironmental factors such as UV light irradiation.

The melanin pigment, which is produced in melanin cells on the bodyskin, is a phenolic polymer having a complex of a black pigment and aprotein. It blocks the sun's ultraviolet rays to protect the skin organsunder the dermis and, at the same time, removes free radicals generatedin skin tissues so as to protect proteins and genes in the skin.However, melanin, produced by internal or external stress stimuli in theskin, is a stable substance, which is not removed even when the stressesdisappear, until it is discharged to the outside by skin keratinization.Thus, when melanin is produced in an unnecessarily large amount,hyperpigmentations, such as discoloration, freckles and spots, which areunfavorable in terms of beauty, will occur. As people who like outdooractivity have increased with an increase in leisure population, the needto prevent melanin pigmentation caused by UV light has increased. Inorder to satisfy this need, ascorbic acid, kojic acid, albutin,hydroquinone, glutathione, or derivatives thereof, or substances havingtyrosinase inhibitory activity, have been used in cosmetics or medicaldrugs. However, the use thereof has been limited due to insufficientwhitening effects and various problems, such as skin safety problems,and formulation and stability problems which occur when they are addedto cosmetics.

DISCLOSURE Technical Problem

Accordingly, the present inventors have conducted many studies toprepare whitening cosmetic composition which is derived from naturallyoccurring substances, is safe for the skin and, at the same time, has anexcellent whitening effect and excellent product stability. As a result,the present inventors have found that ginsenoside Fl obtained byallowing plant extract, particularly ginseng extract, to react with anacid, an alkali or an enzyme, has an excellent effect of reducingmelanin biosynthesis, thereby completing the present invention.

Therefore, it is an object of the present invention to examine themelanin biosynthesis inhibitory effect of ginsenoside Fl obtained byallowing plant extract to react with an acid, an alkali or an enzyme andto provide a cosmetic and therapeutic composition having an excellentwhitening effect.

Technical Solution

The present invention provides ginsenoside Fl represented by thefollowing formula 1 and the use of a cosmetic composition containing thesame for skin whitening:

Hereinafter, the present invention will be described in detail.

The ginsenoside Fl is obtained from a plant extract. Particularly, theginsenoside Fl which is contained in the cosmetic composition of thepresent invention is obtained from ginseng, red ginseng, white ginseng,fresh ginseng, ginseng tails, ginseng leaves or ginseng berries.

The extract that is used in the present invention may be prepared in thefollowing manner. An organic solvent is added to a plant in an amount ofabout 1-6 times, and preferably about 3 times the weight of the plant,and the plant is defatted by extracting it 1-5 times at roomtemperature. An organic solvent is added to the defatted plant in anamount of about 1-8 times, and preferably about 4 times the weight ofthe defatted plant, and the defatted plant is extracted 1-5 times underreflux. The extract is incubated at 10-20° C. for 1-3 days, and thenseparated into residue and a filtrate through filtration andcentrifugation. The separated filtrate is concentrated under reducedpressure, and the concentrate is suspended in water, and then treatedwith, for example, ethanol, to remove pigments therefrom. Then, theaqueous layer is extracted 1-5 times with an organic solvent, and thenthe obtained organic solvent layer is concentrated under reducedpressure to obtain an organic solvent extract. The organic solventextract is dissolved in a small amount of methanol or the like, and thena large amount of ethyl acetate is added thereto. The producedprecipitate is dried, thus obtaining the extract of the presentinvention. The organic solvent that is used in the present invention ispreferably either a single solvent selected from the group consisting ofanhydrous methanol, hydrated methanol, anhydrous ethanol and hydratedethanol, or a mixed solvent of one of the above-mentioned methanols andone of the above-mentioned ethanols.

The extract is hydrolyzed using an acid, a base, an enzyme or amicroorganism producing the enzyme, thus preparing ginsenoside Fl.

When the acid is used, it may be either at least one acid selected fromthe group consisting of hydrochloric acid, sulfuric acid and nitricacid, or a mixture of said acid with at least one alcohol selected fromthe group consisting of ethanol, methanol and butanol. The alcohol thatis used in the acid-alcohol mixture is preferably 50% ethanol. To theextract, either a 0.1-2N acid solution (preferably a 1N acid solution)or the acid-alcohol mixture, is added, and then the extract ishydrolyzed by heating under reflux in a water bath at a temperature of50-100° C. (preferably 80° C.) for 0.5-2 hours.

When the base is used, it may be either at least one base selected fromthe group consisting of sodium hydroxide and potassium hydroxide, or amixture of said base with at least one alcohol selected from the groupconsisting of ethanol, methanol and butanol. The alcohol that is used inthe base-alcohol mixture is preferably 50% butanol. To the extract,either a 0.1-2N base solution, preferably a 1N base solution, or thebase-alcohol mixture, is added, and then the extract is hydrolyzed byheating under reflux in a water bath at a temperature of 50-100° C.(preferably 100° C.) for 1-12 hours.

When the enzyme is used, it may be at least one selected from the groupconsisting of β-glucosidase, α,β-arabinosidase, α,β-rhamnosidase,β-glucuronidase, β-galactosidase and amyloglucosidase. The extract isdissolved in a 5-20-fold volume (preferably about 10-fold volume) of anacidic buffer solution, and then the enzyme is added thereto. Then, theextract is stirred in a water bath at about 37° C. for about 1-60 hoursand, at the same time, the elimination rate of the substrate is examinedby thin layer chromatography. When the substrate is completelyeliminated, the extract is heated in hot water at 80-100° C. for 5-15minutes. Then, the hydrolysis reaction is terminated and the reactionsolution is collected.

When the microorganism is used, it may be a microorganism producing saidenzyme. More specifically, the microorganism may be selected from thegroup consisting of aspergillus sp., bacillus sp., penicillium sp.,rhizopus sp., rhizomucor sp., talaromyces sp., bifidobacterium sp.,mortierella sp., Cryptococcus sp., microbacterium sp., etc. The extractis dissolved in a 5-10-fold volume (preferably an about 10-fold volume)of ionized water, and then it is sterilized at 121° C. for 30 minutesand cooled to 30° C. Precultured microorganisms are inoculated into theextract solution in an amount of 5-10 wt % based on the weight of theextract solution and cultured at 30° C. for 2-5 days. Then, thescavenging rate of the substrate is examined by thin layerchromatography, and when the substrate is completely eliminated, thereaction is completed, and the reaction solution is centrifuged at5,000-10,000 rpm. The collected precipitate is washed three times withdistilled water, thus obtaining a precipitate.

After the hydrolysis reaction is carried out as described above using anacid, a base, an enzyme or a microorganism producing the enzyme, thereaction solution is concentrated under reduced pressure to remove thesolvent. To the residue, an alcohol selected from the group consistingof methanol, ethanol and butanol is added, and the solution is stirred1-5 times. Then, the precipitated salts are removed by filtration, andthe filtrate is concentrated under reduced pressure, thus obtainingginsenoside Fl.

The whitening cosmetic composition of the present invention containssaid ginsenoside Fl in an amount of 0.0001-10 wt % based on the totalweight of the composition. If the content of ginsenoside Fl is less than0.0001 wt %, it cannot provide a whitening effect and the like, and ifthe content exceeds 10 wt %, the increase in the content will not leadto an increase in the effect thereof.

The whitening cosmetic composition of the present invention inhibits theproduction of skin melanin induced by UV light or inflammation andameliorates pigmentation.

There is no particular limitation on the formulation of the whiteningcosmetic composition of the present invention, and the inventivecomposition can be formulated into cosmetic products, for example, skinlotion, astringent lotion, milk lotion, nourishing cream, massage cream,essence, eye cream, eye essence, cleansing cream, cleansing foam,cleansing water, pack, powder, body lotion, body cream, body oil andbody essence.

ADVANTAGEOUS EFFECTS

The ginseng-derived ginsenoside Fl(20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) which is providedaccording to the present invention has the effects of inhibiting melaninproduction and ameliorating pigmentation. Because of such effects, theginsenoside Fl (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) may beadvantageously used in cosmetic compositions for inhibiting melaninproduction and ameliorating pigmentation produced by UV light.

BEST MODE

Hereinafter, the present invention will be described in further detailwith reference to examples and test examples, but the scope of thepresent invention is not limited only to these examples.

Reference Example 1 Preparation of Ginseng Extract

2 kg of ginseng was added to 4 l of aqueous methanol solution andextracted 3 times under reflux, followed by incubation at 15° C. for 1day. Then, the extracted plant was separated into residue and a filtratethrough filter-cloth filtration and centrifugation, and the filtrate wasconcentrated under reduced pressure. The concentrate was suspended inwater, and then extracted five times with 1 l of ethanol to removepigments therefrom, and the aqueous layer was extracted three times with500 ml of 1-butanol. The resulting total 1-butanol layer wasconcentrated under reduced pressure to obtain a 1-butanol extract whichwas then dissolved in a small amount of methanol and added to a largeamount of ethyl acetate. The produced precipitate was dried, thus 100 gof a ginseng extract.

Example 1 Preparation of Ginsenoside Fl by Acid Hydrolysis

10 g of the ginseng extract obtained in Reference Example 1 was added toa 20-fold volume (v/w) of a 1N HCl-50% methanol solution (v/v) andhydrolyzed by heating under reflux in a water bath at 80° C. for 8hours. Then, the reaction solution was concentrated under reducedpressure to remove the solvent, and the residue was added to 200 ml ofethanol and stirred three times. Then, the precipitated salts wereremoved by filtration, and the filtrate was concentrated under reducedpressure, thus obtaining a crude product. The crude product was purifiedby silica gel column chromatography (chloroform:methanol=8:1-4:1), thusobtaining 1.25 g of ginsenoside Fl.

Example 2 Preparation of Ginsenoside Fl by Base Hydrolysis

10 g of the ginseng extract obtained in Reference Example 1 wasdissolved in 500 ml of dry pyridine, and 10 g of sodium methoxide powderwas added thereto. Then, the solution was hydrolyzed by heating underreflux in a water bath for 8 hours, and the reaction solution wasconcentrated under reduced pressure to remove the solvent. The residuewas added to 200 ml of ethanol and stirred three times. The precipitatedsalts were removed by filtration. The filtrate was concentrated underreduced pressure, thus obtaining a crude product. The crude product waspurified by silica gel column chromatography(chloroform:methanol=8:1-4:1), thus obtaining 0.85 g of ginsenoside Fl.

Example 3 Preparation of Ginsenoside Fl by Enzyme Hydrolysis

10 g of the ginseng extract obtained in Reference Example 1 wasdissolved in 100 ml of a 0.1M acetic acid solution (pH 4.5), and 2.5 gof enzymes (0.5 g of hesperidinase, 0.5 g of naringinase, 0.5 g ofcellulose, 0.2 g of β-glucuronidase, and 0.3 g of amyloglucosidase;manufactured by Sigma) was added. Then, the solution was stirred in awater bath at 37° C. for 48 hours and heated in hot water at 80-100° C.for 10 minutes. After completion of the reaction, the reaction solutionwas concentrated under reduced pressure to remove the solvent, and theresidue was added to 200 ml of ethanol and stirred three times. Theprecipitate was removed by filtration, and the filtrate was concentratedunder reduced pressure, thus obtaining a crude product. The crudeproduct was purified by silica gel column chromatography(chloroform:methanol=8:1-4:1), thus obtaining 0.91 g of ginsenoside Fl.

Example 4 Preparation of Ginsenoside Fl Using Microorganisms

10 g of ginseng extract obtained in Example 1 was dissolved in 100 ml ofionized water and sterilized at 121° C. for 30 minutes, followed bycooling to 30° C. Then, precultured Aspergillus niger KCCM 11885 wasinoculated into the extract solution in an amount of 5-10 wt % based onthe weight of the extract solution and cultured at 30° C. for 5 days.Then, the elimination rate of the substrate was examined by thin layerchromatography, and when substrate was completely eliminated, thereaction was completed. The culture was centrifuged at 5,000-10,000 rpm,and the collected precipitate was washed three times with distilledwater, and then centrifuged to collect the reaction solution as aprecipitate. Then, the precipitate was added to 200 ml of ethanol andstirred three times. Then, the precipitate was removed by filtration,and the filtrate was concentrated under reduced pressure, thus obtaininga crude product. The crude product was purified by silica gel columnchromatography (chloroform:methanol=8:1-4:1), thus obtaining 0.62 g ofginsenoside.

Test Example 1 Measurement of Melanin Production Inhibitory Effect UsingMouse Melanocytes

Mouse melanocytes (Mel-Ab cells) derived from C57BL/6 mice were culturedin DMEM (Dulbecco's modified Eagle's media), containing 10% fetal bovineserum, 100 nM 2-O-tetradecanoyphorbol-13-acetate and 1 nM cholera toxin,in conditions of 37° C. and 5% CO₂. The cultured Mel-Ab cells weredetached using 0.25% trypsin-EDTA and cultured in a 24-well plate at aconcentration of 10⁵ cells/well. For 3 days from 2 days after the startof culture, 10 ppm of each of test substances (kojic acid, ReferenceExample 1 and Examples 1 to 4) was added to the cells being cultured.Herein, kojic acid was used as a control group in order to examine themelanin production inhibitory effect of ginsenoside Fl of the presentinvention.

The medium was removed, and the cells were washed with PBS and lysedwith 1N sodium hydroxide. The absorbance of the cells was measured at400 nm, and then the melanin production inhibitory rate (%) of each testsample was calculated according to the resulting equation 1. Thecalculation results are shown in Table 1 below (Dooley method).

Melanin production inhibitory rate (%)=100−(absorbance of each testsample/absorbance of control group×100)  [Equation 1]

TABLE 1 Melanin production inhibitory effect Test samples Melaninproduction inhibitory rate (%) Reference Example 1 21.3 Example 1 35.1Example 2 33.6 Example 3 34.2 Example 4 34.9 Kojic acid 30.5

As can be seen in Table 1 above, the ginsenoside Fl according to thepresent invention showed excellent melanin production inhibitory rate(%) compared to the existing whitening agent kojic acid. In addition, itcan be seen that the melanin production inhibitory rate (%) was almostsimilar between the hydrolysis methods of preparing the ginsenoside Flfrom the ginseng extract.

Test Example 2 Test of Whitening Effect on Human Skin

An opaque tape having a perforated hole of a 1.5 cm-diameter wasattached to the upper arm of each of 12 healthy men. Then, UV rays (UVB)were irradiated to each of the subjects at a dose 1.5-2 times higherthan the minimal erythema dose, thus inducing skin's blackness.

After the UV irradiation, a 1 wt % solution of each of the test samples(1,3-butyleneglycol: ethanol=7:3, as a vehicle) was applied to the skinof each subject. As negative and positive control groups, a 3 wt %solution of each of kojic acid and a vehicle was applied to the skin ofeach subject. A control group was not applied with anything, and achange in the state of the subject's skin was observed for 10 weeks. Theskin color was measured with a colorimeter (Minolta, Japan) at a 1-weekinterval. Then, the difference (ΔL*) in skin color between the timepoint of application and the time point of completion of application ofeach sample was calculated according to the following equation 2, andthe calculation results are shown in Table 2 below. Meanwhile, thewhitening effect of each sample was evaluated by comparing ΔL* betweenthe site applied with each sample and the control site. In theevaluation, a ΔL* value of about 2 indicates that the pigmentation isclearly ameliorated, and a ΔL* value higher than about 1.5 indicatesthat the sample has a whitening effect.

ΔL*=L*value at time point of completion of application−L*value at timepoint of start of application  [Equation 2]

TABLE 2 Whitening effect on human skin Test samples Lightless (ΔL*) ofskin color Control group 0.45 ± 0.13 Vehicle 0.50 ± 0.15 Kojic acid 1.56± 0.11 Reference Example 1 1.26 ± 0.13 Example 1 1.60 ± 0.13 Example 21.67 ± 0.17 Example 3 1.71 ± 0.13 Example 4 1.64 ± 0.21

As can be seen in Table 2, the ginsenoside Fl according to the presentinvention had a ΔL value higher than 1.6, suggesting that it had anexcellent whitening effect. Furthermore, it can be seen that theginsenoside Fl according to the present invention had an excellentwhitening effect compared to kojic acid known to have an excellentwhitening effect.

INDUSTRIAL APPLICABILITY

As described above, the ginsenoside Fl extracted from ginseng hasexcellent effects of inhibiting melanin production and amelioratingpigmentation produced by UV light. Accordingly, the present inventioncan provide a cosmetic composition, which contains the ginsenoside Fl asan active ingredient, and thus has an excellent whitening effect.

1.-11. (canceled)
 12. A method of whitening skin of a subject byapplying to the skin a cosmetic composition containing ginsenoside Fl(20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) as an activeingredient.
 13. The method of claim 12, wherein the ginsenoside Fl ispresent in an amount of 0.0001-10 wt % based on the total weight of thecomposition.
 14. A method of inhibiting melanin production in a subjectby applying to the skin a cosmetic composition containing ginsenoside Fl(20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) as an activeingredient.
 15. The method of claim 14, wherein the ginsenoside Fl ispresent in an amount of 0.0001-10 wt % based on the total weight of thecomposition.
 16. A method of ameliorating skin pigmentation produced byUV light in a subject by applying to the skin a cosmetic compositioncontaining ginsenoside Fl(20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) as an activeingredient.
 17. The method of claim 16, wherein the ginsenoside Fl iscontained in an amount of 0.0001-10 wt % based on the total weight ofthe composition.
 18. A cosmetic composition containing ginsenoside Fl(20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) obtained from anextract of ginseng, red ginseng, white ginseng, fresh ginseng, ginsengtails, ginseng leaves or ginseng berries.
 19. The cosmetic compositionof claim 18, wherein the ginsenoside Fl is obtained by allowing theextract to react with an acid, a base, an enzyme or a microorganismproducing the enzyme.
 20. The cosmetic composition of claim 19, whereinthe acid is either at least one selected from the group consisting ofhydrochloric acid, sulfuric acid and nitric acid, or a mixture of theacid with at least one alcohol selected from the group consisting ofethanol, methanol and butanol.
 21. The cosmetic composition of claim 19,wherein the base is either at least one base selected from the groupconsisting of sodium hydroxide and potassium hydroxide, or a mixture ofthe base with at least one alcohol selected from the group consisting ofethanol, methanol and butanol.
 22. The cosmetic composition of claim 19,wherein the enzyme is at least one selected from the group consisting ofβ-glucosidase, α,β-arabinosidase, α,β-rhamnosidase, β-glucuronidase,β-galactosidase and amyloglucosidase.
 23. The cosmetic composition ofclaim 18, wherein the microorganism is at least one selected from thegroup consisting of aspergillus sp., bacillus sp., penicillium sp.,rhizopus sp., rhizomucor sp., talaromyces sp., bifidobacterium sp.,mortierella sp., Cryptococcus sp., and microbacterium sp.